Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), is a serious agricultural pest in China. Previous studies have shown that olfaction plays a crucial role in the survival and reproduction of herbivorous insects, such as A. lineolatus, and OBPs (odorant-binding proteins) are involved in the initial steps of olfaction. Due to the complex intracellular environment in insects, detection of the expression of OBP genes by conventional methods is limited in vitro. In this study, we developed a PNA-GO- (peptide nucleic acid and graphene oxide) based mRNA biosensor that allows detection of the expression of the AlinOBP14 gene in A. lineolatus. We demonstrate that AlinOBP14 is expressed in the adult head, which is consistent with previous research on the expression profiles of AlinOBP14 in A. lineolatus using quantitative real-time PCR. The results demonstrate that the method we developed is both feasible and reliable. In addition, this method exhibited good stability and specificity in detection of chemically synthesized targets in vitro as well as the expression of the target OBP gene in vivo.The alfalfa plant bug, A. lineolatus, the physiological functions of Plus-C OBPs remain largely unknown. In addition, detection of the expression of insect OBP genes by conventional methods is difficult in vitro. Here, we focused on AlinOBP14, a Plus-C OBP from A. lineolatus, and we developed a PNA-GO-based mRNA biosensor to detect the expression of AlinOBP14. The results demonstrated that AlinOBP14 plays dual roles in A. lineolatus. The AlinOBP14 is expressed beneath the epidermis of the vertex and gena in heads of A. lineolatus, and it functions as a carrier for three terpenoids, while AlinOBP14 is also expressed in the peripheral antennal lobe and functions as a carrier for endogenous compounds such as precursors for juvenile hormone (JH) and JHⅢ. Our investigation provides a new method to detect the expression of OBP genes in insects, and the technique will facilitate the use of these genes as potential targets for novel insect behavioral regulation strategies against the pest.OBPs play a crucial role in the recognition of ligands and are involved in the initial steps of semiochemical perception. The diverse expression of OBP genes allows them to participate in different physiological functions in insects. In contrast to classic OBPs with typical olfactory roles in  Olfaction, an important chemosensory modality in insects, plays a crucial role in sensing chemical signals from the environment ,2. InsecAdelphocoris lineolatus (Goeze) (Hemiptera: Miridae), has become a serious pest in transgenic Bt cotton fields in China [A. lineolatus, including the classic OBP subfamily and the Plus-C OBP subfamily [AlinOBP14 gene, a Plus-C OBP gene from A. lineolatus. The aims of this study were to identify the physiological roles of AlinOBP14 in A. lineolatus and to facilitate the implementation of the OBP gene as a potential target for designing behavior control strategies to effectively manage the pest.The alfalfa plant bug, in China ,7,8. Thiubfamily . In the At present, real-time quantitative PCR (RT-qPCR) is widely used for analyzing relative expression levels of OBP genes in insects ,11,12. AGO is a single-atom-thick carbon material and a water-soluble derivative of graphene due to the presence of suspended hydroxyl and carboxylic groups at the surface . Single-AlinOBP14 gene in A. lineolatus  to detect chemically-synthesized target mRNA in vitro and the expression of the AlinOBP14 gene in A. lineolatus. The biosensor we developed allows monitoring of three regions of the target mRNA in A. lineolatus without cross-reactivity. We found that AlinOBP14 is highly expressed in the adult head of A. lineolatus, consistent with previous research on the expression profiles of AlinOBP14 in A. lineolatus by quantitative real-time PCR [A. lineolatus, we conclude that the method we developed can be applied to detect the expression of mRNA in insects.In this study, we report a PNA-GO-based mRNA biosensor that allows simple, specific detection of the expression of the time PCR . The resGO  was purchased from XFNANO Materials Tech Co., Ltd. . The mRNA used in the study was synthesized by Sangon Biotech Co., Ltd. . Each PNA probe was labeled with a different fluorescent dye; these were obtained from Sangon Biotech Co., Ltd. . The probes were purified by HPLC. All PNA and mRNA sequences are listed in Supporting Information A. lineolatus adults were collected from cotton fields at the Lang Fang Experimental Station of the Chinese Academy of Agricultural Sciences, Hebei Province, China. In the laboratory, the colony was reared on green beans (Phaseolus vulgaris L.) and 10% sucrose solution. The laboratory colony was kept in plastic containers (20 cm × 12 cm × 8 cm) and maintained at 30 °C, 60 ± 5% relative humidity (R.H.), with a 14:10 light: dark (L:D) photoperiod [The toperiod . Fourier transform infrared (FT-IR) measurement of GO powder was carried out with a NICOLET IS10 FTIR spectrometer  by the KBr pellet method (GO:KBr = 1:100). Raman spectra were obtained using a DXR Raman Microscope  with an excitation wavelength of 514 nm. The X-ray diffraction (XRD) patterns were recorded on a Bruker D8 X-ray diffractometer . Atomic force microscopy (AFM) images were obtained by using a SPM 9700 microscope . The samples were prepared by depositing dispersions of GO on a freshly cleaved mica surface. The images were flattened and analyzed using the Gwyddion software. In this assay, 15 µL of Cy5-P1 (10 µM), 80 µL of ROX-P2 (10 µM), 100 µL of FAM-P3 (10 µM) were mixed with 650 µL of HEPES buffer , followed by the addition of 8 µL of GO aqueous solution (1 mg/mL) and reacted at room temperature for 1 min. After that, the fluorescence measurements were carried out on a FL6500 fluorescence spectrometer . In the spectral scan mode, the excitation wavelength of Cy5-P1 was 643 nm, and emission was monitored from 650 to 750 nm; for ROX-P2 these were 575 nm and 580–640 nm; FAM-P3 used 494 nm and 520–550 nm. The mixture of three PNA probes  and GO nanocomplex was prepared as a PAN-GO-based mRNA biosensor.The hybrid nano-complex solution (PNA-GO-based mRNA biosensor) was mixed with chemically synthesized AlinOBP14-T1, AlinOBP14-T2, and AlinOBP14-T3, respectively, followed by addition of various concentrations (0–1000 nM) of target mRNA and reacted at room temperature. Then, fluorescence measurements were carried out using a FL6500 fluorescence spectrometer  to monitor the hybridization process. For the operation in the spectral scan mode, the excitation and emission wavelengths of three PNA probes were the same as those in the preparation of the mRNA biosensor. A mixture of hybrid nano-complex (PNA-GO-based mRNA biosensor) was added to solutions consisting of different combinations of three targets , respectively. All of the concentrations of the targets were 1000 nM. The mixtures were reacted at room temperature for 12 h. As a control, the PNA-GO-based mRNA biosensor was added with chemically synthesized mRNA with a random sequence (1000 nM) and reacted at room temperature for 12 h. Then, fluorescence intensities corresponding to FAM, ROX, and CY5 were measured using a FL6500 fluorescence spectrometer . In the spectral scan mode, the excitation and emission wavelengths of the PNA probes were same as those used in the preparation of the mRNA biosensor.A. lineolatus adult using a PLI-100 pico-injector  manipulated by a MP-255 micromanipulator  under a microscope. Each treatment was applied to 10 A. lineolatus adults , and non-injected A. lineolatus adults  were used as a control.The PAN-GO nano-complex solution (100 nL) was injected at the conjunction between the prothorax and mesothorax of each AlinOBP14 gene in antennae, stylets, the heads without antennae and stylets, thoraces, abdomens, legs, and wings from injected and non-injected A. lineolatus adults of both genders was observed by a Zeiss LSM 880 confocal microscope. In brief, the different tissues of injected and non-injected A. lineolatus were embedded in Tissue-Tek optimal cutting temperature (O.C.T.) compound  and cut into 12 µm slices at −20 °C by using a freezing microtome . Sections were collected on superfrost plus microscope slides . The slides were dried at room temperature and fixed in 4% paraformaldehyde solution at 4 °C for 30 min, incubated in 0.2 M HCl for 10 min, and washed in PBS buffer. The slides were mounted using ProLongTM Glass antifade mountant . The tissues were observed using a Zeiss LSM880 confocal microscope . Images and colocation characteristics of enhanced fluorescent signals were processed with ZEN 3.2 .After 12 h, the expression of the A. lineolatus adults were cleared following the PEGASOS recirculation procedure [A. lineolatus adults were anesthetized and immersed in 4% paraformaldehyde (PFA) at room temperature for 12 h before proceeding to tissue clearing; the adults were then immersed in 20% EDTA (PH 7.0) at 37 °C in a shaker for four days . The samples were washed with H2O for at least 30 min to elute excessive EDTA. Following this, samples were decolorized with the quadrol decolorization solution for two days and placed in 5% ammonium solution at 37 °C in a shaker. Samples were placed in gradient tB delipidation solutions for 1–2 days and then in tB-PEG for two days for dehydration. Samples were then immersed in the BB-PEG medium at 37 °C for at least one day for clearing.In order to observe the sensilla on the epidermis of the vertex and gena and the structure of antennal lobes, the whole body of non-injected rocedure . The maiThe chemically synthesized GO was water dispersible. We first characterized the material as prepared GO (1 mg/mL) sheets to confirm their exfoliation using Fourier transform infrared (FT-IR), Raman, X-ray diffraction, and Atomic force microscopy (AFM). The analyses revealed that the average thickness of GO was ~1 nm, characteristic of a fully exfoliated GO sheet. All of the analysis data indicated the successful preparation of GO  and GO nano-complex was prepared as a PAN-GO-based mRNA biosensor.To prepare the PNA-GO-based mRNA biosensor, we studied the adsorption properties of PNA probes on the GO surface and monitored the quenching kinetics by measuring the fluorescence changes of the dye-labeled PNA probes. Then, we optimized the relative amounts of the dye-labeled PNA probes and GO to give more than 97% quenching of the fluorescence by measuring fluorescent spectra of the PNA probes in the presence of GO. The results showed that the addition of 8 µL of GO (1 mg/mL) resulted in 97% fluorescent quenching of 15 µL of CY5-P1 (10 µM), 80 µL of ROX-P2 (10 µM), and 100 µL of FAM-P3 (10 µM) within 1 min (Supporting Information AlinOBP14) as the targets, the PAN-GO-based mRNA biosensor was mixed with chemically synthesized AlinOBP14-T1, AlinOBP14-T2, and AlinOBP14-T3 at various concentrations (0–1000 nM), respectively. The results showed that the fluorescent intensity gradually increased within a range of 0–1000 nM of target mRNA . Thus, it was necessary to design multiple probes to simultaneously monitor different regions of the target mRNA. In this study, we designed three PNA probes conjugated with three different fluorescent dyes complementary to the selected regions of target mRNA . Then, we evaluated the sequence specificity and multiplexed sensing capability of the PNA-GO-based mRNA biosensor in a homogeneous solution. The mRNA biosensor was treated with solutions consisting of different combinations of the three targets. The fluorescence intensity corresponding to Cy5-P1 was increased when mRNA biosensor was mixed with chemically synthesized AlinOBP14-T1, indicating that the PNA probe conjugated with Cy5 hybridized with AlinOBP14-T1 . However, the full-length mRNA sequence of OBP14-T1 a1–a3. ThOBP14-T1 b1–b3. ThOBP14-T1 c1–c3. ThOBP14-T1 d1–d3. ThOBP14-T1 e1–e3. ThOBP14-T1 f1–f3. ThOBP14-T1 g1–g3. CoOBP14-T1 .A. lineolatus. The PAN-GO nano-complex solution (100 nL) was injected into A. lineolatus adults. Non-injected A. lineolatus adults were used as a control. After 12 h, the different tissues of A. lineolatus adults were cut into 12 µm slices. Fluorescent images of the cells in A. lineolatus were obtained using a Zeiss LSM 880 confocal microscope.Due to the complex intracellular environment, it is not trivial to simultaneously detect multiple targets in insects, and therefore none of the previously reported mRNA biosensors have been employed for multiplexed targets sensing in insects. We examined whether the PAN-GO-based mRNA biosensor could detect the three selected regions of the target mRNA  in A. lineolatus adults . The female adults were used in the following study unless otherwise indicated. Images of the cells in A. lineolatus showed that the intracellularly delivered PAN-GO-based mRNA biosensor induced an increase of the fluorescence intensity beneath the epidermis of the vertex and gena in the head of adult A. lineolatus  recirculation procedure. The antennal lobe in the head of A. lineolatus is a sphere-shaped part of the deutocerebrum  PNA vantages ; (2) Thevantages ; (3) PNAvantages ; (4) PNAvantages . A. lineolatus. Moreover, we found several sensilla located at the epidermis of the vertex and gena in A. lineolatus adults. Several divergent Drosophila OBPs are expressed in olfactory and gustatory sensilla [AlinOBP14 is expressed in the cells beneath the epidermis of the vertex and gena in the head of adult A. lineolatus. To verify this hypothesis, the enhanced fluorescence signals beneath the epidermis of the vertex and gena of A. lineolatus were used for colocalization analysis. The results indicated that CY5-P1, ROX-P2, and FAM-P3 were located beneath the epidermis of the vertex and gena. Therefore, three PNA probes hybridized with corresponding complementary target mRNA beneath the epidermis of the vertex and gena, and AlinOBP14 was expressed in the sensilla beneath the epidermis of the vertex and gena of A. lineolatus. In sensilla, ligands enter the sensillum lymph to interact with receptor molecules located on the neuronal dendrites, OBPs functions as carriers for binding and transport of ligands, and they protect chemical ligands from enzymatic modification in the sensillum lymph. AlinOBP14 was detected beneath the epidermis of the vertex and gena. Is AlinOBP14 related to olfactory recognition? Fluorescence-based competitive binding assays showed that three terpenoids, including β-ionone, nerolidol, and trans, trans-farnesol strongly bind to AlinOBP14 [AlinOBP14 functions as a carrier for the three terpenoids.In this study, we found that the intracellularly delivered PAN-GO-based mRNA biosensor induced an increase in the fluorescence intensity beneath the epidermis of the vertex and gena in the head of adult sensilla . Generalsensilla . It has linOBP14 . Those tlinOBP14 ,45,46. Ttrans, trans-farnesol is not only a plant volatile but also an endogenous compound that functions as a precursor for insect JH biosynthesis [AlinOBP14 [AlinOBP14 also related to bind and transport insect JH in A. lineolatus? In this study, we also found that the intracellularly delivered PAN-GO-based mRNA biosensor induced an increase of the fluorescence intensity in the peripheral antennal lobe, suggesting that AlinOBP14 is expressed in the peripheral antennal lobe. To verify this hypothesis, the enhanced fluorescence signals in the peripheral antennal lobe were used for colocalization analysis. CY5-P1, ROX-P2, and FAM-P3 were detected in the peripheral antennal lobe. This suggested that the three PNA probes hybridized with corresponding complementary target mRNA in the peripheral antennal lobe, indicating expression of AlinOBP14. What is the connection among AlinOBP14, JH, and the antennal lobe? Previous studies have shown that JH can affect honey bee behavioral development by altering levels of the neuromodulators in the antennal lobe [AlinOBP14 is also expressed in the peripheral antennal lobe of A. lineolatus and functions as a carrier for the endogenous compounds, including precursors for JH and JHIII. However, these hypotheses require experimental testing, for example via monitoring the expression level of AlinOBP14 in the head of A. lineolatus after stimulation with these four compounds. Although this method can be used to transduce the complex process of the AlinOBP14 gene expression into visual images and allows simple, specific detection of the expression of the AlinOBP14 gene, the quantitative monitoring of mRNA expression level of AlinOBP14 cannot be performed, as the PNA probes hybridize with target mRNA in A. lineolatus at room temperature over a 12 h period, and the expression level of AlinOBP14 gene will decreased when the PNA probes hybridizes with target mRNA.In addition, ynthesis ,48, and linOBP14 . Is Alinnal lobe  and can nal lobe ,51,52. TAlinOBP14 gene in A. lineolatus. PNA probes were adsorbed onto GO, and thus could be loaded onto GO and delivered through the cell membrane to hybridize with target mRNA in living cells without nuclease- or protease-mediated degradation. Furthermore, the mRNA biosensor had sufficient selectivity to differentiate complementary and non-complementary targets. Additionally, the PNA probes hybridized only with complementary mRNA in a sequence-specific manner and simultaneously sensed multiple targets in a homogeneous solution without cross-reactivity. However, the quantitative monitoring of mRNA expression levels in insects still needs to be combined with conventional methods, such as Northern blotting and real-time qPCR.In this study, we found that the PNA-GO-based mRNA biosensor enabled monitoring of the expression of the AlinOBP14 is expressed beneath the epidermis of the vertex and gena and in the peripheral antennal lobe in the head of adult A. lineolatus. This study is the first to detect the expression of a Plus-C OBP gene in A. lineolatus using the mRNA biosensor. Furthermore, this method could be used to provide a theoretical basis for pest management strategies based on manipulating insects away from food or interrupting their behavior, for example, attractants and repellents could be screened out from the ligands for use in pest control. In addition, we expect that the PNA-GO-based mRNA biosensor can be applicable in olfactory-related research in insects. For example, the detection of the expression of OBP genes in insects and monitoring of the expression of olfactory receptor (OR) genes would facilitate research concerning the molecular basis for olfactory reception of insects. This would in turn aid implementation of the olfactory related genes as pest control targets via interference with pest olfactory recognition [Overall, the present study shows ognition .AlinOBP14 in A. lineolatus. AlinOBP14 was expressed beneath the epidermis of the vertex and gena, and in the peripheral antennal lobe. Our previous work showed that β-ionone, nerolidol, trans, trans-farnesol, and insect JHIII strongly bind to AlinOBP14. Thus, we propose that AlinOBP14 plays a dual role in the recognition of ligands, not only functioning as a carrier for terpenoids but also as a carrier for endogenous compounds. We designed a PNA-GO-based mRNA biosensor to detect chemically synthesized target mRNA in vitro and the expression of


Microcystin-LR (MC-LR) exists widely in polluted food and water in humid and warm areas, and facilitates the progression of colorectal cancer (CRC). However, the molecular mechanism associated with the MC-LR-induced CRC progression remains elusive. The purpose of this study is to explore the role of the hub genes associated with MC-LR-induced CRC development at the molecular, cellular and clinical levels through bioinformatics and traditional experiments. By utilizing R, we screened and investigated the differentially expressed genes (DEGs) between the MC-LR and the control groups with the GEO, in which, HOXB4 highly expressed in MC-LR-treated group was identified and further explored as a hub gene. With the aid of TCGA, GEPIA, HPA, UALCAN, Cistrome, and TIMER, the increased mRNA and protein levels of HOXB4 in CRC tissue were found to be positively associated with high tumor stage and poor prognosis, and were linked to immune infiltration, especially tumor-associated macrophages and cancer-associated fibroblasts. Cox regression analysis and nomogram prediction model indicated that high HOXB4 expression was correlated to poor survival probability. To elucidate the mechanism of high HOXB4 expression induced by MC-LR, we overlapped the genes involved in the MC-LR-mediated CRC pathways and the HOXB4-correlated transcription genes. Importantly, C-myc instead of PPARG and RUNX1 promoted the high expression of HOXB4 through experiment validation, and was identified as a key target gene. Interestingly, C-myc was up-regulated by HOXB4 and maintained cell cycle progression. In addition, MC-LR was proved to up-regulate HOXB4 expression, thus promoting proliferation and migration of Caco2 cells and driving the cell cycle progression. In conclusion, MC-LR might accelerate CRC progression. In the process, MC-LR induced C-myc augmentation elevates the high expression of HOXB4 through increasing the S phase cell proportion to enhance Caco2 cell proliferation. Therefore, HOXB4 might be considered as a potential prognostic biomarker for CRC. Microcystins (MCs) are the largest and most diverse group of cyanotoxins . Of overColorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related mortality worldwide . Generalvia DHDDS (Homeobox B4 (HOXB4) belongs to the homeobox (HOX) family that possesses transcription factor activity and is essential for stem cell self-renewal and tumorigenesis . It has ia DHDDS . Howeveria DHDDS . Bhatlekia DHDDS , 16. Meaia DHDDS . HoweverIn this work, microarray data analyses and experiment validation were implemented to comprehensively determine the pathways and hub genes that most likely participated in MC-LR-induced CRC progression. Our findings demonstrated that cell cycle progression was dependent on the promotion of C-myc, and that C-myc was regulated by MC-LR-induced high HOXB4 expression. Therefore, MC-LR enhanced CRC cell proliferation. Moreover, HOXB4 was linked to poor prognosis in CRC after covering a group of factors including gene expression, survival status, clinical-pathological parameters, immune infiltration, the biological function, and relevant cellular pathway.The gene expression profiles of MC-LR-exposed CRC cells by using GSE29861 dataset were obtained from GEO database . The GSEP<0.05 and [logFC]>1.0 were considered as the cut-off criteria. The heatmap, UMAP, volcano plot and box plot were conducted by using “Complex Heatmap”, “umap” and “ggplot2” packages of R software.The normalized expression matrix of microarray data was downloaded from the GSE29861 dataset, and the probes corresponding to multiple molecules were then removed. The “limma” package of R software was used to identify DEGs across experimental condition, and the significant DEGs with https://genome-cancer.ucsc.edu/) to analyze the expression level of HOXB4 in colon adenocarcinoma (COAD), as well as its expression level of messenger RNA (mRNA). To obtain the data on protein immunohistochemical analysis of tumor tissue and normal human tissue, the Human Protein Atlas online website (https://www.proteinatlas.org/) was used. Besides, the online pathology database provided protein-specific expression and location information for 48 normal tissue samples, 20 typical categories of cancer, 47 cell lines and 12 blood cells.We utilized The Cancer Genome Atlas (TCGA) project (https://david.ncifcrf.gov/) is a crucial foundation for the success of any high-throughput gene annotation, integrated discovery function and visualization analysis, which provides gene biological meaning for analyzing the function and pathway enrichment of DEGs  online tools, which are newly developed web-based tools providing key interactive and customizable function based on TCGA and genotype tissue expression data. According to the median of HOXB4 gene expression, cut off-low (50%) and cut off-high (50%) values were used as the expression thresholds for dividing the patients into two groups, namely the low HOXB4 expression group and the high HOXB4 expression group.To verify the prognostic values of HOXB4 in COAD patients, we obtained the significant data of HOXB4 on overall survival (OS) and disease free survival (RFS) of tumors derived from TCGA analyzed with GEPIA2 (test) and UALCAN , and progression free interval (PFI) of the patients in two cohorts. We used the multivariate analysis to assess whether HOXB4 was an independent prognostic factor for CRC patient survival. The HOXB4 expression level was considered statistically significant in Cox regression analysis when For ascertaining the expression of HOXB4 in cancer prognosis, we built a nomogram which was considered as an effective and convenient method for predicting the OS, DSS, and PFI of CRC patients in the TCGA cohort , 23. Thehttp://cistrome.org/TIMER/) web server (via the TISIDB database (http://cis.Hku.hk/TISIDB/)  values were calculated by means of the purity-adjusted Spearman’s rank correlation test.To determine the association between the HOXB4 expression level and immune infiltration according to the TCGA tumor data, we used the Tumor Immune Estimation Resource 2.0 (TIMER2.0)  . The P-vhttp://cistrome.org/db), which is a resource of human and mouse cis-regulatory information derived from DNase-seq, ChIP-seq, and ATAC-seq chromatin profiling assays, to obtain the information on gene regulatory analysis. Moreover, we overlapped the genes which were regulated by HOXB4 involved in CHEA Transcription Factor Targets Dataset and Cistrome cancer Dataset. Afterwards, we performed the R package of “cluster Profiler” to automate the results of the enrichment analysis . The RT-qPCR kit was purchased from TAKARA, and RT-qPCR was performed with the CFX96 real-time system . The primers were designed by Sangon Biotech Co., Ltd. , and the list of sequences is shown in Proteins (30-50 μg) were separated on 12% sodium dodecyl sulfate polyacrylamide gels and were then transferred to polyvinylidene fluoride membranes  at 4°C. The membranes were blocked at 37°C for 1 h and were then incubated overnight with the primary antibodies at 4°C followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive proteins were detected using the enhanced chemiluminescence system (Amersham-Pharmacia Biotech) and subsequent autoradiography.2. The cells were treated with different doses of MC-LR  for 24, 48, or 72 h. The control cells were cultured with 0.1% DMSO.The human colorectal adenocarcinoma cell line Caco2 was obtained from the American Type Culture Collection  and synchronized by 24 h of serum deprivation. The cells were grown in DMEM with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified atmosphere of 5% COCells were incubated in a 96-well plate with 3000 cells per well. Then, 10 μl of cell counting kit-8 reagent  was added to each well for 1 h, and the optical density (OD) value was detected using a Thermo Scientific Multiskan MK3 .Cells were cultured in six-well plates until they reached 100% confluence. A vertical or horizontal wound was gently created in the monolayer using a 20 μl sterile pipette tip. The cells were then washed 3 times with growth medium to remove the detached cells, and fresh medium was added. The images were captured using an inverted microscope and camera at the designated times, and wound closure was assessed using Image Pro Plus 6.0. The rate of wound healing = [(the wound width of 0 h -t h)/0 h wound width] *100% (t: 24/48/72 h).Caco2 cells were pretreated with MC-LR at different concentrations  for 24 h in 24-well plates. Then, 600μL of supernatant was gathered and affiliated into the RAW264.7 cells. Besides, the cell proliferation reagent CCK-8 was applied to approximately measure the RAW264.7 cell viability.5 RAW264.7 cells in a volume of 100μL were co-cultured in the upper chambers . Then, Caco2 cells were cultured in lower chambers and pretreated with MC-LR 5μM.To establish the co-culture system between Caco2 and RAW264.7 cells, 2 × 105 cells were cultured in the 8 μm well  for 24 hour and fixed in paraformaldehyde for 15 min, then stained with crystal violet for 10 min. Cells migrate through the well were counted in microscope.2 × 10Cells in each group were fixed in 75% ethanol for 24 h at -20°C. After treated with 50 μg/ml RNase  for 30 min at 37°C, the cells were stained with 50 μg/ml propidium iodide  for 15 min, then analyzed by BD Accuri c6 , and Flow JO software.post hoc test was performed. P-values < 0.05 were regarded as significant.Statistical analyses were performed with GraphPad Prism 8.0 . For two-group comparisons, two-sided unpaired Student’s t-test was performed. For multiple comparisons, one-way followed by Dunnett’s P=0.045) than in normal tissue in the TCGA  (P=0.047) (P=0.046)  of COAD presented patients categorized in the HOXB4-higher-score group had a significantly worse RFS  and the validation set (P=0.013) , pathologic stage, and age were negative predictors for OS and DSS in COAD patients with univariate analysis , CD4+ T cells , macrophages , neutrophils , dendritic cells  and tumor purity in COAD, but no association was observed with B cells   , cellular component (CC), and molecular function (MF), to show the main possible routes of the MC-LR in the Caco 2 cells. As shown in in vitro. Caco2 cells were treated with various doses of MC-LR  for 24, 48, or 72 h. CCK-8 assay indicated that the 5 µM of MC-LR significantly promoted the proliferation of Caco2 cells after treatment for 24 h. Subsequently, the 10 µM of MC-LR also remarkably improved the proliferation of Caco2 cells after treatment for 48 h and 72 h . With aid of using TISCH , the UMAP and bar charts reflected the cellular composition of the tumor microenvironment in CRC samples  in COAD after the application of the GEPIA (P=0.046) . In addition, HOXB4 is a key regulator of NK cell function, and its application in the generation of functional NK cells with increased lytic potential may be of great significance for cancer immunotherapy. We adopted multiple immune deconvolution methods to find a statistically positive correlation of HOXB4 expression with B cells, macrophages, CD8+ T cells, CD4+ T cells, dendritic cells, neutrophils, and cancer-associated fibroblasts  for 24h were found to increase the expressions of multidrug resistance genes MRP1 and MDR1 in HepG2 cells . Howeverin vivo. Therefore, In the following future research, we would like to enlarge the sample size of CRC patients to provide a better understanding of the relationship between the exposure level of MC-LR and CRC. Meanwhile, studying the association of MC-LR exposure and HOXB4 levels with tumor stage and prognosis of CRC and exploring the underlying molecular mechanism would be beneficial as well. In addition, we will perform xenograft assay to demonstrate the underlying mechanisms by which MC-LR promotes colorectal cancer development, and further provide the clue in the status of immune infiltration to MC-LR-caused colorectal cancer progression.However, our study has several limitations. First, bioinformatics analysis was not coupled with our own collected clinical sample. Second, we were not sure the interaction between the immune infiltration and CRC cells in the presence of MC-LR In summary, we screened that the HOXB4 was a molecular target of MC-LR in promoting CRC progression with the aid of GEO. Our analyses showed statistically significant correlations of HOXB4 expression with prognosis, immune cell infiltration, constructing protein interaction networks and enrichment analysis of HOXB4-related transcription genes. Furthermore, we indicated the MC-LR induced C-myc augmentation elevates the high expression of HOXB4 through increasing the cell cycle progression to enhance Caco2 cell proliferation. These analysis results may aid the insight to the role of HOXB4 in MC-LR mediated-CRC progression from the perspective of clinical tumor sample studies.The original contributions presented in the study are included in the article/LW contributed to conception and design of the study, wrote the first draft of the manuscript. HJ organized the database. YZ wrote sections of the manuscript and organized the database. JW wrote sections of the manuscript. WC, YT, and KC organized the database and performed the statistical analysis. ZQ and WF performed the statistical analysis. ZZ contributed to conception and design of the study, administrated project, wrote review &edited. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by the Natural Science Foundation of Chongqing (Grant No. cstc2019jcyj-msxmX0632) and the National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hormonal contraceptive (HC) use has been associated with an increased risk of developing a depressive episode. This might be related to HC’s effect on the serotonergic brain system as suggested by recent cross-sectional data from our group, which show that healthy oral contraceptive (OC) users relative to non-users have lower cerebral serotonin 4 receptor (5-HT4R) levels. Here, we determine if cerebral 5-HT4R binding differs between HC non-users, OC users, and hormonal intrauterine device (HIUD) users among women with an untreated depressive episode. Also, we test if antidepressant drug treatment response and its association with pre-treatment 5-HT4R binding depends on HC status.11C]-SB207145 Positron Emission Tomography imaging data from the NeuroPharm-NP1 Study (NCT02869035) were available from 59 depressed premenopausal women, of which 26 used OCs and 10 used HIUDs. The participants were treated with escitalopram. Treatment response was measured as the relative change in the Hamilton Depression Rating Scale 6 items (rΔHAMD6) from baseline to week eight. Latent variable models were used to evaluate the association between global 5-HT4R binding and OC and HIUD use as well as rΔHAMD6.[6; OC users had 3-4% lower binding compared to non-users for every 10% percent less improvement in HAMD6. Within the groups, we observed a trend towards a positive association in non-users (padj=0.10) and a negative association in OC users (padj=0.07). We found no strong evidence of a difference in treatment response between the groups (p=0.13).We found no evidence of a difference in global 5-HT4R binding between depressed HC users and non-users (p≥0.51). A significant crossover interaction (p=0.02) was observed between non-users and OC users in the association between baseline global 5-HT4R binding and week eight rΔHAMDvs. non-users in depressed women, however, it seemed that 5-HT4R settings differed qualitatively in their relation to antidepressant drug treatment response between OC users and non-users. From this we speculate that depressed OC users constitutes a special serotonin subtype of depression, which might have implications for antidepressant drug treatment response.We found no difference in 5-HT4R binding between HC users  Hormonal contraception (HC) is used by millions of women worldwide to avoid pregnancies as well as for other indications such as dysmenorrhea, menorrhagia, and acne , 2. In s11C]SB207145-radioligand positron emission tomography (PET)) to quantify the postsynaptic serotonin 4 receptors (5-HT4R). The 5-HT4Rs are sensitive to chronic synaptic serotonin manipulation such that they are inversely correlated to serotonergic tonus , hormonal intrauterine devices (HIUDs), vaginal rings, injections and subdermal implants . OCs andic tonus –25 makinic tonus . In compic tonus . The SSRic tonus , 26. Frovs. those using OCs and HIUDs, 2) if antidepressant drug treatment response is affected by HC use, 3) if an association between pre-treatment 5-HT4R brain binding and antidepressant drug treatment response depends on HC status, and 4) if neostriatal 5-HT4R levels are sensitive to SSRI treatment in HC users. We hypothesize that if the effects from OC and MDD are not additive, we will not be able to detect a difference in baseline 5-HT4R binding. However, when we account for treatment response, and thus may account for the serotonergic subtype of MDD, we will be able to detect a negative main effect of OC use on 5-HT4R binding. In line with that, we hypothesize that an association between baseline binding and treatment response will depend on OC use, such that an association is found in non-users but not in OC users, as they may constitute a more homogenous group in terms of 5-HT4R downregulation. We further hypothesize that the effect of HIUD use on 5-HT4R binding will be in the same direction as of OC use, but with smaller effect size, due to the smaller degree of synthetic steroid exposure and larger degree of preserved ovulations .We sought to investigate this by determining 1) if the 5-HT4R binding differs between depressed women who do not use HC vs. those who do not use OC ) depressive episode confirmed by a psychiatrist at inclusion. The current depressive episode was single or recurrent and did not involve acute severe suicidal ideation or psychosis. They had no prior or present history of other major psychiatric disorders confirmed by use of the diagnostic tool, the Mini International Neuropsychiatric Interview version 6.0  from the NeuroPharm-NP1 study with available baseline 5-HT4R PET data and information on HC use. One patient was excluded from the analyses on 5-HT4R imaging data due to an interrupted PET scan . Eight patients dropped out during follow-up, six OC users and two non-users . Twenty-six of the remaining women were rescanned at follow-up; 15 were non-users, six were OC users and five were HIUD users . The women were suffering from an unmedicated moderate to severe  and progestogen-only pills (n=6), as specified in Information about HC use was acquired 6) in percentage from baseline to week eight. In the larger NeuroPharm-NP1 study, the categorical treatment response categories, remitters vs. non-responders, were used as the primary clinical outcome and rΔHAMD6 was included as the secondary outcome  were quantified using the simplified reference tissue model with cerebellum as reference tissue , we used the Kruskal-Wallis Rank Sum Test. For reporting the effect size, we used the Mann-Whitney parameter (ND and week eight rΔHAMD6 depends on OC/HIUD use (aim 3), we extended our LVM by including an interaction term between rΔHAMD6 and the HC group to be mediated through the global LV. The estimates related to rΔHAMD6 are expressed as the percent change in 5-HT4R BPND per 10% change in HAMD6 (r10ΔHAMD6). To evaluate the effect of eight weeks of SSRI/SNRI treatment on neostriatal 5-HT4R binding across groups (aim 4), we used linear regression adjusting for the difference in injected [11C]SB207145 mass per kg bodyweight between baseline and week eight scans. Due to the smaller re-test samples within the OC and the HIUD group, the p-values were also computed with a permutation test with 10,000 permutations.To evaluate if HC use was associated with global 5-HT4R brain binding (aim 1), we used a linear latent variable model (LVM) where the effects of HIUD- and OC use were mediated through a shared latent variable  across brain regions of low (neocortex), intermediate (hippocampus) and high (neostriatum) 5-HT4R binding . By intrarameter , which gDiagnostic tools were used to assess the adequacy of the models’ assumptions. We used chi-squared tests to evaluate specification of the covariance structure for each LVM with chi-square < 0.05 indicating suboptimal specification [section 6.2.4 in ]. Missin6/17) at baseline. The depressive symptoms scored within a moderate-to-severe depressive episode as restricted by the inclusion criteria. The educational level tended to differ between the groups (p=0.06) such that the HC users tended to have lower education. As expected, the OC users had lower plasma estradiol levels  compared to the non-users , padj=0.0002, and the HIUD users , padj=0.01. The OC users had lower plasma progesterone levels but only compared to the non-users (padj=0.04). The HIUD users did not seem to differ from the non-users in plasma hormone levels.The clinical profile and PET parameters of the study population at baseline are shown in adj>0.79), so no additional covariance was added to the model. There was no evidence for an association between 5-HT4R binding and OC use (p=0.51) or HIUD use (p=0.73) . From the chi-squared test we found no evidence for a lack of fit (p(p=0.73) Figure 16) between the groups (p=0.13).The estimated probability of finding a smaller reduction in HAMD6 in an OC and an HIUD user was 66%  and 68% , respectively, compared to in a non-user (adj=0.04) and the HIUD users (padj=0.01); 77.8% of the OC users were treated with the highest recommended daily dose of 20 mg compared to 35.0% and 22.2% of the non-users and the HIUD users, respectively. Correspondingly, the OC users also had a higher plasma concentration of escitalopram at week eight compared to the HIUD users (padj=0.03), and at a trend level compared to the non-users (padj=0.06). The dropouts’ depression profiles, in terms of baseline HAMD6/17 as well as relative change in HAMD6 in percentage from baseline to week one, two, four and eight and reason for dropout, are presented in Week eight depression profiles are shown in non-user Figure 2adj>0.41). The estimated associations between baseline global 5-HT4R binding  and treatment responses across each group are shown in -2 (p=0.10)), a trend towards a negative slope in the OC users (-1.27x10-2 (p=0.07)), and a non-significant negative slope in the HIUD users (-3.34x10-4 (p=0.75)). The corresponding correlation coefficients are 0.39 for the non-users, -0.46 for the OC users, and -0.12 for the HIUD users. In the OC users, the slope differed by -2.59x10-2 (p=0.02) from the non-users. In 10ΔHAMD6 mediated through the global LV in the non-users (p=0.10). The second layer (B) shows how this association differs in the HC users from the non-users. The regional estimates show that per 10% less improvement in HAMD6, the OC users had 2.6-4.0% (padj ≤ 0.03) lower binding at baseline compared to the non-users. The third layer (C) shows the estimated effect of HC use on 5-HT4R binding if no treatment response was achieved at week eight, i.e., the estimated HC effect when accounting for the confounding factor of a suggested serotonergic subtype of MDD. However, as we have only few observations at this part of the response scale, these estimates are calculated based on the model and less on actual observed differences. At the regional level, the percent effect of OC use varied between -24.2% and -16.3% (padj ≤ 0.01). No gross deviation from the normality assumption was observed for any of the LVMs.Again, the LVM was supported by the region-specific bindings loading onto the global LV (p<0.001). The chi-squared test showed no evidence of misspecification of the covariance structure . Due to the small sample sizes in the HIUD- and the OC group, the effect sizes are less reliable, and the p-values are also computed after 10,000 permutations. It revealed that, if the null hypothesis is true , the observed lower 5-HT4R BPND would be found by chance in 9-10% of the cases, hence, a reduction in 5-HT4R binding is only found at a trend level in the HIUD and the OC users.In ND and treatment response in the non-users and the OC users and these were in opposite directions with a significant crossover interaction, such that the OC users had 3-4% lower binding compared to the non-users for every 10% percent less improvement in HAMD6 at week eight. Based on our model, the main effect of OC use on regional 5-HT4R binding at zero improvement in depressive symptoms was estimated to be between -16 and -24%. In addition, we observed no strong evidence of a difference in treatment response between the groups, although, the estimated probability of finding a smaller reduction in HAMD6 in an OC and an HIUD user was numerically higher compared to in a non-user. Last, we found that as in the non-users, both the HIUD users and the OC users seemed to react to SSRI treatment in terms of neostriatal 5-HT4R downregulation, however, only at a trend level.In this study, we found no evidence of a difference in 5-HT4R binding in unmedicated depressed women who use OCs or HIUDs relative to non-users. We found a trend towards an association between baseline global 5-HT4R BPIn a healthy population we found OC use to be associated with 9-12% lower global 5-HT4R binding , which wND and treatment response, but notably, the association within each group was only borderline significant, which may be due to power issues. As we were not expecting to see a negative trend in the association in the OC users, this highlights the question whether the 5-HT4R setting may differ qualitatively, i.e., whether the mechanisms behind a lower 5-HT4R level differ. As shown in a mixed male-female cohort of MDD patients, only those responding to treatment had a lower baseline (unmedicated) 5-HT4R level compared to healthy controls, so it has been speculated whether they have a serotonergic subtype of MDD, in which the lower binding is due to a compensatory upregulation of the serotonergic tonus as an attempt to maintain euthymia , we compare the effect of OC use with a population with a non-serotonergic subtype of MDD, which reveals an OC effect equal to about -20%. However, since this result is based on our model with rather few observations in this range of the treatment response, it should be interpreted with some caution.Whereas we found no difference in 5-HT4R binding level at baseline between the groups, we observed a crossover interaction between the OC users and the non-users in the association between baseline 5-HT4R BPeuthymia . In conteuthymia . This aleuthymia , 42, redeuthymia , increaseuthymia  and increuthymia , which mnd -beta . Also nond -beta . We specThe association between baseline binding and treatment response did not differ between the HIUD users and the non-users, however, the estimate was in the same direction as in the OC users. It is possible that, if replicated in larger sample sizes, this reflects an effect of HIUD use on serotonin brain signaling.6 in an OC and an HIUD user compared to a non-user was 66% and 68%, respectively, but the confidence intervals included 50%, so we have insufficient evidence to conclude that differences in the treatment response exist. This might be a question of power as the confidence intervals were wide, e.g., the OC users vs. the non-users 95% confidence spanned from 48% to 80%. Larger samples would help narrow the confidence intervals to help us discriminate between differences in treatment response vs. no or very small differences. The median rΔHAMD6 was, at the non-significant level, about 18 percent point and 14 percent point lower in the OC and the HIUD users, respectively, relative to the non-users. This is equivalent to about two points based on median HAMD6 scores at baseline. Treatment response could be affected by educational level . Limited and insufficient clinical evidence exists addressing whether HC use affects SSRI treatment response. The STAR*D study reported a trend towards better remission rates to citalopram treatment in 226 HC users compared to 670 HC non-users, however, this effect was not robust to adjustment for potential confounders  the sample size is small, so interpretation should be made with caution, especially with regard to HIUD use and when comparing treatment responses, 2) the OC group was pooled from users of different types of OCs (combined OCs and progesterone-only pills) with different hormone content, which may blur the results, 3) we lacked information on previous HC use among the non-users and information on starting day and pill-free days in the HC users, which could have helped us understand the potential role of HC use in their depressive episode, 4) we had 8 dropouts during follow-up which were mainly OC users. However, based on their last response rate Table S2vibe.frokjaer@nru.dk) or access can be requested through the procedures outlined here: https://cimbi.dk/index.php/documents/category/3-cimbi-database.The data analyzed in this study is subject to the following licenses/restrictions: For accessing the dataset, a Cimbi database application is required. Requests to access these datasets should be directed to the corresponding author . The patients/participants provided their written informed consent to participate in this study.SL and VF contributed to study conceptualization, data collection, analysis and interpretation of results, participated in drafting the manuscript, and approved the final version prior to submission. BO contributed to analysis and interpretation of data, revised the manuscript critically for important intellectual content, and approved the final version prior to submission. KK-F, AP, VD, and CS contributed to data collection and the acquisition of data, revised the manuscript critically for important intellectual content, and approved the final version prior to submission. GK and MJ contributed to study conceptualization and interpretation of data, revised the manuscript critically for important intellectual content, and approved the final version prior to submission. All authors contributed to the article and approved the submitted version.The study was funded by Independent Research Fund Denmark (grantID: 0134-00278B & DFF-6120-00038), the Research Fund of the Mental Health Services - Capital Region of Denmark, the Innovation Fund Denmark, H. Lundbeck A/S (grantID: 5189-00087A), the Research Council of Rigshospitalet, the Augustinus Foundation (grantID: 16-0058), and Savværksejer Jeppe Juhl og hustru Ovita Juhls Mindelegat. None of the funders had any influence on study design, data collection, analysis, and result interpretation.VF and GK declare to have received honorarium for advisory meetings at Sage Therapeutics, GK at Sanos, and VF for a symposium lecture at Lundbeck Pharma A/S. MJ has given talks sponsored by Boehringer Ingelheim and H. Lundbeck.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Extracorporeal membrane-oxygenation (ECMO) has been widely used for COVID-19-related acute respiratory distress syndrome (ARDS), with a mortality rate of 37.1% based on the largest published series , includist, 2020, through May 31st, 2021. ECMO in Germany was provided in 213 intensive care units (ICUs)—out of a total of 1.684 intensive care units across 1.288 hospitals (www.intensivregister.de). The study was approved by the Ethics Committee of the Witten/Herdecke University.We report unbiased and unselected follow-up data at hospital discharge from the federal German hospital payment institute (InEK) of all 3.397 COVID-19 patients supported with VV-ECMO in Germany from March 1The mean age of all ECMO patients remained stable over the study period 57 ± 11 years). As expected, survivors were younger than the non-survivors (53 years (range 47–63 y) vs. 59y (Range: 54-63y)), independent of timing during the pandemic. The mean duration of ECMO support was not different between survivors and non-survivors, averaging 17 days. Overall, in-hospital mortality was 68%. Of note, the mortality for any given week  cohort with a mortality of 51.9% in patients initiated on ECMO after May 1, 2020 . A comprA major strength of the study is inclusion of every COVID-19 patient treated with VV-ECMO in Germany  over the course of the study. One limitation is the lack of granularity of the data, including comorbidities, impairing our ability to better understand the reasons for the high mortality.Ultimately, the present data should serve as a warning to clinicians. Even in a country with adequate resources, mortality in ECMO-supported patients with COVID-19-related respiratory failure may be high if its use is not restricted to patients deemed most likely to benefit.


Alternaria alternata (Alt). The mice were exposed to Alt extract for twelve hours or ten days to induce allergic airway inflammation. C57BL/6 mice exposed to Alt responded with eosinophilic infiltration and the characteristic IL-5 upregulation. In contrast, the inflammatory response to Alt extract in BALB/c mice was characterized by a neutrophilic response, high levels of G-CSF, and elastase in the lungs. The lack of neutrophils affected the processing of IL-33 in BALB/c mice, as was demonstrated by depletion of neutrophils through intraperitoneal injections of anti-Ly6G antibody. Our data identifies the key role of neutrophils in airway inflammation through IL-33 cleavage in the Alt-induced airway inflammation in mice, which could potentially underline the different endotypes in human disease.Future precision medicine requires further clarifying the mechanisms of inflammation in the severe endotypes of chronic airway diseases such as asthma and chronic rhinosinusitis (CRS). The presence of neutrophils in the airways is often associated with severe airway inflammation, while their precise contribution to the severe inflammation is largely unknown. We aimed to study the role of neutrophils in BALB/c and C57BL/6 mice exposed to  Alt plays an important role in the proteolytic cleavage of IL-33FL into a shorter mature form (IL-33103-270)  with nasal polyps remain the most severe and often uncontrolled extremes of type 2 inflammatory diseases of the upper and lower airways , 2. Type103-270) , 23. In and BALF . The regof IL-33 , 24. Thead libitum and were kept in a 12-hour/12-hour light/dark cycle. Seven-weeks old female BALB/c or C57BL/6J wild-type mice  were used in the study. Mice were lightly anesthetized with isoflurane/air  when receiving applications. For the acute model of airway inflammation, one intratracheal (i.t.) application of 20 µg of Alt extract  in 50 µl PBS or 50 µl PBS alone  was given to the mice. Twelve hours after the application, the mice were euthanized with an intraperitoneal (i.p.) injection of 150 µl Dolethal . For the Alt-induced asthma model, mice were first sensitized with an i.t. application of 5 µg Alt extract in 50 µl PBS or 50 µl PBS and seven days later, mice were challenged every day for three times with 20 µg Alt extract in 50 µl PBS or 50 µl PBS as earlier described . Murine BALF was collected by flushing the airways as described before  and perfMurine lungs were homogenized with the TissueLyser LT . T-Per Tissue Protein Extraction Reagent and HALT protease inhibitor cocktail kit (ThermoFisher Scientific) were used. 25 μg total protein of lung homogenate was loaded to a 4 – 15% Mini-PROTEAN TGX Stain-Free Gels . The proteins were separated by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). For immunostaining, the primary antibody anti-mouse-IL-33  and secondary antibody anti-goat-horseradish peroxidase  were used, together with anti-β-actin  and anti-mouse-HRP (ThermoFisher Scientific). The visualization was performed with Immobilon Western Chemiluminescence HRP substrate  and measured with the Chemidoc system (Bio-Rad). The band intensities were semi-quantitatively analyzed with Fiji  by measuring the area under the peak.Formalin-fixed paraffin-embedded lung tissue was cut at 4 µm. The sections were stained for goblet cells using the Periodic Acid-Schiff kit (Sigma-Aldrich) conform the manufacturer’s instructions. The number of positive cells in the larger airways, with a perimeter of 600 to 2000 μm, were counted and normalized to the perimeter of airways measured using ImageJ software.Murine IL-4, IL-5, IL-13, IL-17, IL-25, IL-33, G-CSF and GM-CSF were measured in lung homogenates using the Mouse Magnetic Luminex assays (R&D Systems) conform the manufacturer’s protocol. Mouse TSLP and elastase were measured with the Quantikine ELISA kit (R&D Systems). Murine cathepsin G and proteinase 3 were measured with ELISA .Data analysis was performed with Prism 9 . The normality of the data was tested with a D’Agostino & Pearson test. Normally distributed data were analyzed using a one-way ANOVA with a Tukey’s test as correction for multiple comparisons, while not normally distributed data were analyzed using a one-way ANOVA Kruskal-Wallis test with a Dunn’s test as correction for multiple comparisons. Data analysis was performed with Prism 9 .Alt extract  was significantly more pronounced in the lungs, while in C57BL/6 mice also full-length IL-33  was present  application of hallenge , 28. RemAlt extract and then challenged with 20 µg of Alt extract i.t. on days 7, 8, and 9 . IL-33 protein levels were higher in the lungs of BALB/c mice after Alt treatment, and a trend for an increase in C57BL/6 could be observed  with anti-Ly6G neutralizing antibodies or an isotype control antibodies prior to a single i.t. application of Alt extract . Remarkably, this initial innate response persisted after multiple Alt challenges. During sensitization and challenge phases, C57BL/6 mice showed increased levels of IL-5 and a stronger eosinophilic inflammation, while BALB/c mice did not. On the contrary to C57BL/6 mice, BALB/c mice showed higher levels of neutrophils.We have demonstrated that The strain specific difference in mice has been noted before. The ovalbumin treated C57BL/6J mice have a massive influx of eosinophils and neutrophils in the airways, while BALB/c only showed a modest to weak response . At the Alt extract and affect processing of IL-33FL in the lungs.In conclusion, in the current study we extend our knowledge on the regulatory mechanisms of IL-33 in the airway inflammation. Our study demonstrates that different inbred mouse strains become a useful tool for studying the endotypes of inflammatory airway diseases with BALB/c mice showing a mixed inflammatory response characterized by neutrophilic inflammation and typical type 2 cytokine profile. The use of different mouse strains is a largely underestimated tool that could offer more insights in understanding the complex interactions of immune cells occurring during chronic airway inflammation in human patients. Neutrophils play a crucial role in control of allergic type 2 immune response towards The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Ethical Committee of Ghent University.OK designed and performed experiments, analyzed data and wrote the original draft. SN performed experiments, analyzed data, wrote the manuscript. JO, GH, NZ, HB, and NR performed experiments and analyzed the data. TM and HB provided analysis tools and revised the manuscript. CB supervised the study, provided analysis tools, and revised the manuscript. All authors contributed to the article and approved the submitted version.The study has been supported by FWO grant 3G065319 to CB and OK, and FWO grant 3G041819 to TM. SN is a doctoral fellow paid by the Fund for Scientific Research Flanders - Strategic Basic Research (3S035019).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, the lower amplitudes were restored during the remission phase. Except for the E wave, A wave and Sep e’, the UCG results showed no significant differences between the groups. Four patients with anti-MDA5 (+) DM had many leads with lower T wave and cardiac abnormalities  on and after admission. Though anti-MDA5 (+) patients clinically improved after immunosuppressive therapy, some of their ECG findings did not fully recover in remission phase. In conclusion, anti-MDA5 (+) DM appears to show cardiac involvement  during the active phase. Further studies are necessary to clarify the actual cardiac condition and mechanism of these findings in patients with anti-MDA5 (+) DM.Anti-melanoma differentiation-associated gene 5 (MDA5) antibody, a dermatomyositis (DM)-specific antibody, is strongly associated with interstitial lung disease (ILD). Patients with idiopathic inflammatory myopathy (IIM) who are anti-MDA5 antibody positive [anti-MDA5 (+)] often experience chest symptoms during the active disease phase. These symptoms are primarily explained by respiratory failure; nevertheless, cardiac involvement can also be symptomatic. Thus, the aim of this study was to investigate cardiac involvement in anti-MDA5 (+) DM. A total of 63 patients with IIM who underwent electrocardiography (ECG) and ultrasound cardiography (UCG) during the active disease phase from 2016 to 2021  were enrolled in the study, and their clinical charts were retrospectively reviewed. The ECG and UCG findings were compared between the anti-MDA5 (+) and anti-MDA5 (-) groups. All anti-MDA5 (+) patients had DM with ILD. The anti-MDA5 (+) group showed more frequent skin ulcerations and lower levels of leukocytes, muscle enzymes, and electrolytes  than the anti-MDA5 (-) group. According to the ECG findings obtained during the active disease phase, the T wave amplitudes were significantly lower for the anti-MDA5 (+) group than for the anti-MDA5 (-) group (I, II, and V4–6 lead;  Idiopathic inflammatory myopathy (IIM) is an autoimmune disease that affects skeletal muscles and various systemic organs, such as the skin, lungs, heart, and joints , 2. CurrThe anti-melanoma differentiation-associated gene 5 (MDA5) antibody, which is an MSA, was first reported to be an anti-clinically amyopathic dermatomyositis-140 (anti-CADM-140) antibody specific to CADM in a Japanese cohort in 2005, which showed a strong association with rapidly progressive interstitial lung disease (ILD) . The antPrevious studies have shown that 6–75% of the patients with polymyositis (PM)/DM have concomitant cardiac disorders  . Written informed consent was obtained from all the patients prior to enrollment.ECG and UCG findings were reviewed at the first admission for remission induction. The typical ECG wave forms were analyzed using the QP-170D ECG Viewer software version 9 . Two cardiologists (Y.K. and E.T.K.) reviewed the ECG and UCG findings. ECG and UCG parameters were then compared between the anti-MDA5 (+) and anti-MDA5 (-) groups.p < 0.05.The differences between the anti-MDA5 (+) and anti-MDA5 (-) groups were analyzed using an unpaired t-test for the continuous variables and a chi-square test for the categorical variables. Data are expressed as the mean ± standard deviation for the continuous variables and as percentages for the categorical variables, unless otherwise noted. All the statistical analyses were performed using the JMP Pro statistical package version 16.0.0 J . The differences were considered statistically significant at a two-tailed Of the 94 enrolled patients with PM/DM/CADM, 63 underwent both ECG and UCG on admission. We divided these patients into an anti-MDA5 (+) group (n = 21) and an anti-MDA5 (-) group (n = 42), the latter of which comprised fourteen anti-ARS (+), five anti-TIF1-γ (+), seven anti-SRP (+), two anti Mi-2(+), one anti Ku (+), one anti NXP2 (+) and twelve MSA-negative patients. All patients in the anti-MDA5 (+) group had CADM; conversely, all patients in the anti-MDA5 (-) group had PM/DM Figure 1p < 0.01). Platelet, AST, ALT, Na, and Cl levels were also significantly lower in the anti-MDA5 (+) group than in the anti-MDA5 (-) group (p < 0.05). On or after admission, four patients in the anti-MDA5 (+) group and six patients in the anti-MDA5 (-) group showed obvious cardiac abnormality .We compared the baseline clinical data between the anti-MDA5 (+) and anti-MDA5 (-) groups Table 1.p < 0.01; aVF and V3, p < 0.05). The T wave amplitude from the aVR lead, which reflects the reverse direction of the myocardial electrical flow from the other leads, was higher for the anti-MDA5 (+) group than for the anti-MDA5 (-) group (p < 0.01). Among the MSA-positive patients , the anti-MDA5 (+) group tended to show a lower T wave amplitude than the patients with ARS, TIF1-γ, and SRP antibody (data not shown).Next, we analyzed the ECG findings for each group. While the electrolyte levels in all patients were within the normal range on admission, patients with anti-MDA5 (+) CADM tended to show low T waves during the active phase of the disease, but T wave restoration during the remission phase Table 2.p < 0.01; II, aVF and V5 p < 0.05). The four anti-MDA 5 (+) patients with obvious cardiac abnormality had six or more leads with low T-wave.According to the Minnesota code classification system, a low T wave is defined as a T wave amplitude/R wave amplitude (T/R ratio) <0.1 , 12. TheBecause ILD was found to have been present as a complication in the anti-MDA5 (+) CADM group at a significantly higher frequency (100%) than in the anti-MDA5 (-) group (45.2%), we conducted an additional analysis to examine whether the incidence of ILD affected the ECG findings. First, we compared ECG parameters between the anti-MDA5 (-) patients with and without ILD . The non ILD group had significantly higher T waves in the III lead, and conversely, significantly lower T waves in the aVL lead. We could not conclude that the presence of ILD was related to the number of lead with low T wave  group, independent of the presence of ILD.p < 0.05) for the anti-MDA5 (+) group than for the anti-MDA5 (-) group. However, the E/e’ ratio was not significantly different between the groups, and no significant differences in the diastolic interventricular septum or left ventricular posterior wall thicknesses were observed; in almost all cases of the anti-MDA5 (+) group, there was normal cardiac function on echo. These results suggest that subclinical damage of the left ventricle exists in patients with anti-MDA5 (+) CADM  CADM . In thisp < 0.01; For the anti-MDA5 (+) group, we investigated whether ECG changes would be reversible after immunosuppressive therapy. During the active phase of the disease, the number of leads in the 12-lead ECG (not including the aVR lead) that showed a low T wave was higher for the anti-MDA5 (+) group than for the anti-MDA5 (-) group  group. Regarding ECG findings, we divided patients into three groups, according to the number of leads with low T wave: Group A, 0–2 leads (N = 7); Group B, 3–5 leads (N = 6); and Group C, 6 or more leads (N = 8). Group C showed lower T wave amplitudes at multiple leads  CADM. The number of leads with low T wave may be associated with some clinical data . Even if anti-MDA5 (+) patients achieve remission, some may still have abnormalities on ECG and UCG parameters and cardiac damage.+ or Ca+ channels and affects the flow of Ca+ or K+  CADM cohort. In this study, we found characteristic subclinical ECG and UCG abnormalities in patients with anti-MDA5 antibodies , suggesting the extent of cardiac damage. Although there were lower levels of electrolytes in the anti-MDA5 (+) group, there were no severe electrolyte abnormalities that could account for the abnormal ECG findings. With respect to the pathophysiology of anti-MDA5 (+) DM/CADM, elevated levels of several cytokines and chemokines, including interleukin (IL)-6, IL-8, IL-10, IP-10, sCD163, YKL-40, and IFN-α, have been reported –17. Sevea+ or K+ . IL-10, a+ or K+ –22. Daila+ or K+ , and inta+ or K+ , 25. ThuOne hypothesis is that myocardial damage is due to pathological autoantibodies. There have been previous reports of pathological autoantibodies as components of heart damage in mouse models  . HoweverWe also speculate that viral infection may be a trigger, with viral-induced myocarditis leading to the development of anti-MDA5 (+) CADM. It has long been suspected that viruses may be involved in the development of PM/DM  –29. The MDA5 is a viral sensor in the cell, which plays an important role in detecting picornaviruses  that cause myocarditis and encephalitis , 36. DurWe observed that the low T waves recorded in the anti-MDA5 (+) group during the active disease phase were reversible after treatment Table 4.In our study, cardiac function was nearly normal for patients with anti-MDA5 antibodies. Some of the UCG parameters  showed significant differences between the groups. In the remission phase, the values of E wave, A wave, and Sep e were not significantly different, likely due to the small sample size; however, as in the active phase, they remained lower in the anti-MDA5 (+) group than in the anti-MDA5 (-) group, suggesting that some cardiac damage may remain even after remission. The E wave represents the early diastolic filling velocity, which is one of the transmitral flow velocity patterns. When the left ventricular diastolic function decreases, left ventricular relaxation is delayed, and the E wave also decreases. Sep e’ is the peak early diastolic mitral annular velocity at the septum . A low SWe assumed that the ECG results for the anti-MDA5 (+) group were affected by the 100% ILD complication rate in this group; therefore, we conducted a sub-analysis to confirm our hypothesis. Even though ILD was present more frequently in the anti-MDA5 (+) group Table 1,Our study has several limitations. First, this was a retrospective study; therefore, we lacked some clinical data for the included patients. A prospective study is needed to confirm our findings. Second, the clinical implications of a low T wave on ECG, and a low E wave, A wave and Sep e’ on UCG, require further assessment. However, our results may provide new insight into whether anti-MDA5 (+) DM/CADM is a multiorgan autoimmune disease. Therefore, further basic investigations are required.In conclusion, patients with anti-MDA5 antibodies often have specific ECG and UCG findings that indicate subclinical heart involvement. As patients with anti-MDA5 antibodies have a poor prognosis if treatment is delayed, ECG may aid in early detection. Therefore, cardiac function screening and follow-ups for this cohort will be important, even if patients show only slight myocarditis or heart failure.ranran@kuhp.kyoto-u.ac.jp.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of Kyoto University Graduate School and Faculty of Medicine . The patients/participants provided their written informed consent to participate in this study.TaM, YK, and RN designed the study. TaM and TS collected the data. TaM, YK, and EK reviewed the ECG and UCG data. TaM and YK analyzed the data. TaM, YK, EK, and RN interpreted the data. TaM and RN wrote the manuscript. All the authors reviewed and approved the manuscript.This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (18K16145 for RN). The funding body had no role in the study design, the collection, analysis, and interpretation of the data, or in the writing of the manuscript.RN has received research grants from Takeda and Medical & Biological Laboratories Co., Ltd., as well as speaker fees outside of the submitted work from Bristol Myers Squibb, Astellas Pharma Inc., Boehringer Ingelheim, Actelion Pharmaceuticals, and Mitsubishi Tanabe Pharma. HY has received a speaker fee from Boehringer Ingelheim. MT has received research grants and speaker fees from AbbVie GK, Asahi Kasei Pharma Corp., Astellas Pharma Inc., Ayumi Pharmaceutical Corp., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Eisai Co., Ltd., Eli Lilly Japan K.K., Pfizer Inc., UCB Japan Co., Ltd., Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Corp., Novartis Pharma K.K., and Taisho Pharma Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


KD value of 95.30 nM, was successfully obtained by screening and optimization. G48nop showed high specificity towards GYM-A. Based on this, a novel aptasensor based on biolayer interferometry (BLI) technology was established in detecting GYM-A. This aptasensor showed a detection range from 55 to 1400 nM (linear range from 55 to 875 nM) and a limit of detection (LOD) of 6.21 nM. Spiking experiments in real samples indicated the recovery rate of this aptasensor, ranging from 96.65% to 109.67%. This is the first study to report an aptamer with high affinity and specificity for the challenging marine biotoxin GYM-A, and the new established aptasensor may be used as a reliable and efficient tool for the detection and monitoring of GYMs in the future.Gymnodimines (GYMs), belonging to cyclic imines (CIs), are characterized as fast-acting toxins, and may pose potential risks to human health and the aquaculture industry through the contamination of sea food. The existing detection methods of GYMs have certain defects in practice, such as ethical problems or the requirement of complicated equipment. As novel molecular recognition elements, aptamers have been applied in many areas, including the detection of marine biotoxins. However, GYMs are liposoluble molecules with low molecular weight and limited numbers of chemical groups, which are considered as “challenging” targets for aptamers selection. In this study, Capture-SELEX was used as the main strategy in screening aptamers targeting gymnodimine-A (GYM-A), and an aptamer named G48nop, with the highest  Donax deltoides), mussels (Modiolus proclivis), oysters (Saccostrea glomerata), and seawater in many countries [Alexandrium ostenfeldii [Alexandrium peruvianum [Karenia selliformis [Gymnodimines (GYMs) are the smallest members of cyclic imines (CIs) in terms of molecular weight. They are characterized as fast-acting toxins, which can accumulate in filter feeding shellfish and may pose a potential risk to human health and the aquaculture industry ,2,3. Beiountries ,5,6. GYMenfeldii   and Kliformis , which aliformis ,12,13,14liformis . Then, nliformis , GYM-B [liformis , 12-methliformis , GYM-C [liformis , GYM-D [liformis , 16-desmliformis . Their r50 of GYM-A were 3, 80, and 100 μg/kg for i.c., i.p., and subcutaneous injection, respectively [Intracerebroventricular injection (i.c.) or intraperitoneal injection (i.p.) of GYM-A or GYM-B caused rapid death in mice. The average LDectively . Poisoniectively . GYM-A wectively ,22,23,24ectively .No incidents related to human GYM poisoning cases have been reported, and GYMs in shellfish are not regulated in the European Union (EU) or other authoritative organizations ,27,28. GTorpedo californica [T. marmorata [The existing detection methods of GYMs, including mouse bioassay (MBA), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and receptor-binding assay (RBA), have certain defects in practice. MBA is a traditional way to detect GYMs in seafood ,34, but ifornica  and T. marmorata . Thus, Garmorata .Aptamers, which are single stranded DNA or RNA oligonucleotides, are perceived as chemical antibodies. They have the ability to bind to specific targets by crimping and folding into specific tertiary structures. Aptamers are screened from DNA or RNA libraries though systematic evolution of ligands by exponential enrichment (SELEX) ,43. As nMarine toxins, which are mainly small molecular compounds, are one of the main factors leading to marine food poisoning . CurrentGYM-A is a challenging target for screening of aptamers as this molecule is liposoluble and contains a limited number of chemical groups, which makes it hard to be modified and immobilized onto solid medium. Besides, the immobilization of GYM-A on solid medium may cause the change in its native structure, which may reduce the success rate of screening. Therefore, Capture-SELEX was used as the main screening method in this study. In this strategy, ssDNA libraries are immobilized onto magnetic beads by complementary base pairing, while GYM-A that contains native structure is in the solution ,65. The 15 ssDNA oligonucleotides were contained in the first library. Polypeptide marine biotoxin microcystin-LR (MC-LR), whose structure is rich in several chemical groups and does not contain spirocyclic imine, was used as the counter target in round 6–12. As shown in The design of the library, primers, and capture oligo referred to the report by Reinemann  Table S. In thisKD value was chosen for further optimization.The enriched ssDNA from round 12 was cloned into pGEM-18T vector and a total of 80 clones were selected randomly and sequenced. The obtained sequences were analyzed by Clustal X 2.0 software  and diviKD value of G48nop (95.30 nM) was about one third of that of G48 (288 nM), which indicated that the primer binding sequences of G48 were not beneficial for the interaction between G48 and its target. Then, we tried to improve the affinity of G48nop by the removal of some stem sequences in the stem-loop structure, based on the predicted secondary structure of G48nop. However, the affinities of the generated G48nors and G48norsj were not improved accordingly ). The results showed that G48nop could form G-quadruplex and the sequence (5’-GGACGGGAGGTTGG-3’) inset in the G48nop sequence was of the highest G-score . The tendency of G48nop to form a G-quadruplex structure was also analyzed by the online QGRS Mapper  assays . The speKon and Kdis value of G48nop were 9.57 × 106 M−1s−1 and 9.12 × 10−1 s−1, respectively, and the KD value was 95.30 nM. Combined with the interaction curve . The on curve A, a fastThe specificity of G48nop was also determined by the BLI technique. According to chemical structures, marine biotoxins can be divided into the following four categories: polypeptide toxins, polyether toxins, alkaloid toxins, and cyclic imine (CI) toxins ,78. The KD value of G48nop to GYM-A (34.50 ± 1.72 nM) was about one third of the KD value (95.30 nM) determined by BLI  of the biological layer as a function of time [BLI is a label-free technology for measuring the interaction between biomolecules by an optical analytical system. Due to its advantages of rapidness, real-time, and low sample consumption, BLI has been gradually used in many areas ,86. Base of time .Firstly, the linear range, LOD, and LOQ of the aptasensor were measured by testing GYM-A samples in the concentration range of 55–14,000 nM. The results showed that the response values increased with the increasing GYM-A concentration B. The remax is the maximum response value, Rmin is the minimum response value, EC50 represents the concentration at which GYM-A reaches half of the maximum response value, and b is the slope of the curve. Then, we analyzed the data by Graphpad Prism 6.0 software  and obtained the following equation:In this equation, R2 of the data was analyzed to be 0.9963. The curve showed an excellent linear detection at 55–875 nM (y = 0.00018x + 0.09403) was fitted with a correlation coefficient R2 of 0.9916. The limit of detection (LOD) was 6.21 nM  and the limit of quantification (LOQ) was 20.72 nM  [a was the standard deviation of the response of the blank samples (n = 20) and b was the slope of the calibration curve. These results show that this aptasensor has a high sensitivity in detecting GYM-A.The correlation coefficient R5–875 nM D, and a  10Sa/b) . Sa was Secondly, the specificity of this aptasensor was assessed by testing eleven different kinds of toxins  chosen from four categories of marine biotoxins. A mixture of these toxins was also tested by this aptasensor E. The coThirdly, the precision of the aptasensor was assessed by repeating the test three times for GYM-A in a range of 55–14,000 nM. The relative standard deviations (RSDs) of inter- and intra-assay ranged from 0.034 to 4.57%, which were all below 5% . These rFourthly, the feasibility of this aptasensor in real samples was also measured. . GYM-A wThe concentration of GYMs in shellfish samples has been reported in several representative studies ,81,90,91Currently, the detection methods for GYMs are mainly based on LC–MS/MS or RBA . AlthougIn this research, the biorecognition element of GYM-A was the inexpensive DNA aptamer, which is easy to synthesize, store, transport, and is stable even in extreme temperature conditions. The BLI-based aptasensor enables the simultaneous detection of seven GYM-A samples in a small volume of 200 μL within 10 min. Apart from the BLI instrument, only the special biosensor tips and common 96-well plates were the main required materials, and the aptamer modified tips could be reused at least twenty times after washing for ~60 s in detection assays . In concKD value of 95.30 nM was obtained. This aptamer showed high specificity for GYM-A, as determined by BLI and MST assays. G48nop was further used to establish a BLI-based aptasensor. This aptasensor exhibited high sensitivity, specificity, and precision in detecting GYM-A. Furthermore, the good recovery rate and repeatability of this aptasensor in detecting GYM-A in water and shellfish samples further confirmed its feasibility in real samples. Although this aptasensor is not suitable for the detection of seawater samples with very low GYM-A concentrations, its sensitivity is sufficient for most shellfish samples. In conclusion, this BLI-based aptasensor established in this work may be used as a sensitive and reliable tool to detect GYM-A, and the obtained aptamer G48nop with high affinity and specificity may be further used to develop into other types of aptasensors to facilitate the detection of GYM-A in the future.So far, there are no reports of aptamers targeting GYM-A or its analogues. In this study, high-affinity aptamers of the challenging small molecule GYM-A were successfully screened by Capture-SELEX. After further optimization, the aptamer G48nop with the highest TM M-270 streptavidin (diameter 2.8 µm) and the Qubit® ssDNA assay kits were purchased from Thermo Fisher Scientic . GoTaqHot® Start Colorless Master Mix was purchased from Promega Corporation . QIAEX® II gel extraction kits were purchased from Qiagen . Super streptavidin (SSA) sensor biosensors were purchased from ForteBio . Selection Buffer  was purchased from Tiandz . Selection buffer was also used in biolayer interferometry (BLI) experiments. All reagents were of analytical grade and were used without further purification or treatment unless specified. All solutions were prepared with ultrapure water.All ssDNA oligonucleotides were synthesized and purified by high-performance liquid chromatography (HPLC) by Sangon Biotechnology Co., Ltd. . Gymnodimine-A (GYM-A), brevetoxin (BTX), spirolide (SPX), and pinnatoxin (PnTX) were purchased from the National Research Council Canada . Okadaic acid (OA), dinophysistoxin (DTX), palytoxin (PLTX), gonyautoxin (GTX), saxitoxin (STX), microcystin-LR (MC-LR), and nodularin-R (NOD-R) were purchased from Taiwan Algal Science Inc. . Dynabeads® 2.0 fluorometer . The Milli-Q water purification system was purchased form Millipore Corp . BLI assays were conducted by a biolayer interferometry instrument . MST assays were conducted by a Monolith NT.115 system . CD assays were tested by a Chirascan spectrometer .The PCR assays were conducted by a PCR instrument . The rotation of tubes was conducted by a four-dimensional rotating mixer . The DNA concentration was quantified by a Qubit10-TGAGGCTCGATC-N30-CAGAAGACACGCCCGACA-3′) was chemically synthesized. The oligonucleotides (88 nt) of this library included constant primer binding sites (18 nt each) flanking both ends, two different random sequences (N10 = 10 nt and N30 = 30 nt), and a specific docking sequence (12 nt) in the middle. Primer binding sites were designed for the binding of corresponding primers. The forward primer F1 was 5′-GGGAGGACGAAGCGGAAC-3′ and the reverse primer LR1 containing a poly adenine tail (A20 = 20 nt A) was 5′-A20-Spacer18-TGTCGGGCGTGTCTTCTG-3′. The modified reverse primer facilitated the separation of the two different-sized ssDNA sequences of PCR products with double strands. The forward primer and an unmodified reverse primer R1 (5′-TGTCGGGCGTGTCTTCTG-3′) were used to amplify the last round library for cloning. The docking sequence in the library enabled the complementary binding of the library to capture oligo (5′-biotin-GTC-Spacer18-GATCGAGCCTCA-3′), which was modified at 5′ end for binding to streptavidin beads.A random Capture-SELEX library named Bank1 . The hybrid was then incubated with streptavidin beads (washed three times in 1000 μL of selection buffer before use) overnight on a four-dimensional rotating mixer.After overnight incubation, the beads were collected by magnetic separation and washed six times in 1000 μL of selection buffer to remove unbound sequences. After that, the beads were incubated with 200 pmol of GYM-A (dissolved in 1000 μL of selection buffer) for 2 h on a four-dimensional rotating mixer at room temperature. The eluted ssDNA obtained by magnetic separation was quantified by a fluorometer and amplified by PCR . Then, the PCR products were separated by 9% denaturizing urea-polyacrylamide gel electrophoresis (urea-PAGE). The small-sized ssDNA as the target fragment was purified by gel extraction, quantified, and used as the secondary library in the next round.The input amount of ssDNA library was 2 nmol for round 1, 300 pmol for round 2–10, and 200 pmol for rounds 11 and 12. The input amount of GYM-A was kept at 200 pmol each round. During the selection, the pressure increased by adding the counter target of MC-LR, increasing the amount and incubation time of MC-LR, and reducing the incubation time of GYM-A. To improve the specificity, MC-LR was employed as a counter target in round 6–12. After six times of washing, the beads were incubated with MC-LR in 1000 μL of selection buffer. Then they were washed three times in 1000 μL of selection buffer before being incubated with the positive target GYM-A. Detail conditions of the selection are summarized in http://www.unafold.org/mfold/applications/dna-folding-form.php (accessed on 10 February 2022)). The folding temperature was set to 25 °C, and the concentrations of Na+ and Mg2+ were set to 100 mM and 2 mM, respectively. Other parameters were set to be the default in mfold.The ssDNA purified in the last round was amplified by primer F1 and R1. Cloning and sequencing of double-stranded PCR products were conducted by Sangon Biotechnology Co., Ltd. A total of 80 sequences in the sequencing results were clustered by Clustal X 2.1 software and divided into several families. The secondary structure and corresponding Gibbs free energy of candidate aptamers were predicted by the web server of mfold .MST assays were applied to confirm the binding affinity and specificity of aptamers and GYM-A. A Monolith NT.115 system was set with 15% excitation power and 40% MST power at 25 °C. The laser off/on times were 5 and 30 s, respectively. Carboxyfluorescein- (FAM-) labeled aptamers and unlabeled GYM-A were prepared in selection buffer supplemented with Tween-20 of a final concentration of 0.02%. Aptamers (200 nM) treated as the procedure in BLI assays were mixed with a titration series of GYM-A in equal volume, and the mixtures were loaded in capillaries. The final concentrations of GYM-A ranged from 0.305 to 10,000 nM. Assays were conducted more than three times and the affinity constant CD assays were applied to determine the conformation of aptamers. A Chirascan spectrometer was applied with 0.05 cm path length quartz cuvettes. The scanning spectrum ranged from 210 to 320 nm with the step size of 1 nm and the integration time of 0.5 s. Each spectrum scan was the average of three duplicates at 20 °C. The molar ellipticity value was excluded and the selection buffer spectrum value was obtained under the same conditions. CD spectra was analyzed by the Pro-Data viewer software. Unlabeled aptamers (5 μM) were prepared in selection buffer and treated as the procedure in BLI assays before testing.Water samples were treated as in the report by Gao et al. . Water s2Cl2, and the CH2Cl2 was evaporated. The obtained residue was dissolved in 10 µL methanol, and then 10 µL methanol containing GYM-A was dissolved in 500 µL selection buffer. The solution was filtered through a 0.45 µm filter.Shellfish samples were purchased from a market and were treated as in the report by Mukherjee et al. . The GYMt-test was used to compare the significance between all of the groups for the various experiments. The probabilities were p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). The samples were tested at least three times per experiment.Statistical analysis was performed by GraphPad Prism 6.0 Software , and the experiment data were shown as the mean ± standard deviation. One way ANOVA was used to analyze the variance and Dunnett’s